Integral membrane proteins such as GPCRs reside in hydrophobic lipid bilayers. Therefore, recombinant expression has to ensure that GPCRs are targeted and correctly inserted into the host membrane system. Purification of GPCRs must be carried out in the presence of detergents to keep receptors in solution. However, most GPCRs are unstable and denature once extracted from the membrane with detergents. Formation of diffracting crystals requires protein-protein contacts mediated by the hydrophilic regions of the receptor. However, the loops connecting the receptor transmembrane helices, essential for forming crystal contacts, are often small. To date, there is no structure available of a GPCR from a recombinant source. This reflects difficulty in (a) overexpressing receptors in functional form, (b) in maintaining functionality during the purification in the presence of detergents, (c) in developing large-scale purification procedures, and (d) in finding conditions that allow efficient formation of crystal contacts.[unreadable] [unreadable] The development and optimization of heterologous expression and large-scale purification of GPCRs requires robust analysis tools to rigorously assess the quality (functionality) of expressed and purified receptors. We chose the rat high-affinity neurotensin receptor for these purposes because of the availability of a hydrophilic, non-sticky radio-ligand (3Hneurotensin) that allows us to determine the amount of functional receptors, not only in membrane-bound form, but also in the detergent-solubilized state. Using neurotensin receptor as a model for methods development, we have established a bacterial expression system for the production of functional, membrane-inserted receptors, and developed a procedure for the purification of fully functional receptors in detergent solution at the 3-milligram or 10-milligram level, using immobilized metal affinity chromatography and a neurotensin column. This provides high-quality receptor protein for crystallization experiments on a regular basis. We have obtained receptor crystals, which diffract to 12 Angstrom, but show high mosaicity. Efforts to improve the crystal quality are on going. Of particular interest are experimental conditions which stabilize neurotensin receptor in detergent solution to allow the formation of better quality crystals.[unreadable] [unreadable] Structural work on GPCRs must not be limited to receptors only, but receptor / G-protein complexes or receptor / arrestin complexes would provide insight into the mechanism of GPCR-mediated signaling. We have characterized the interaction of purified neurotensin receptor with G-proteins (see below), exploring whether receptor monomers and/or dimers are capable of activating G-proteins. Insight from these experiments will be related to crystallization trials.[unreadable] [unreadable] Structural studies need to be complemented by functional analyses. We have addressed the first step in signal transduction across membranes i.e. the interaction of G-proteins with ligand-activated receptors. GPCRs have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We demonstrated by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At higher receptor concentrations, where NTS1 exists as dimer, agonist (neurotensin) binding displays positive cooperativity. We found that NTS1 monomers activate G-protein, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.